Method to reduce face mask and respirator discomfort

ABSTRACT

Wearing a face mask or a filtering facepiece respirator, especially in hot weather for more than one hour, causes discomfort. But, in a viral pandemic, a barrier to filter inhaled and exhaled air is necessary to protect against airborne pathogens. The sensations of heat abstraction, that is, coolness and cold, can be captured by the rational design of optimized molecules. The topical application of these molecules alleviates the discomfort of wearing a face mask covering. Several entities were identified by synthesis and experiment as having the ideal properties for achieving this purpose. The preferred embodiments are certain 1-diisopropyl-phosphinoyl-alkanes described as DIPA-1-7, DIPA-1-8, and DIPA-1-9, collectively referred to herein as “DIPA compounds.” The applicant found that topical delivery of DIPA compounds to the facial skin, especially to the surface of the external nares (nostrils), alleviates face mask discomfort. From these studies, I hypothesize that the absence of cool air dynamics about the nostrils causes face mask discomfort, not just static heat accumulation or excess humidity. The present discovery pertains to pharmaceutical compositions comprising such compounds, and the use of such compounds and compositions for relieving face mask discomfort.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part to U.S. Ser. No. 15/530,976filed Mar. 31, 2017 and U.S. Ser. No. 16/350,559 filed Nov. 30, 2018.

BACKGROUND OF THE INVENTION

Respiratory tract infections caused by microorganisms such as virusesand bacteria are frequent events. The resultant diseases include sorethroat, acute bronchitis, the common cold, and influenza. Infections ofthe nose, sinuses, pharynx, larynx, and the large airways are generallyself-limiting, with irritation and inflammation, but are seldomlife-threatening. The relatively benign viral pathogens are rhinovirus,influenza virus, adenovirus, enterovirus, and respiratory syncytialvirus. About 15% of bacterial pharyngitis is due to Streptococcuspyogenes. Respiratory pathogens create a substantial economic burden onsociety but are considered an acceptable fact of life. By contrast, thenew SARS-CoV-2 virus (abbreviated here as CoV-2), the causative agent ofthe coronavirus pandemic of 2019+(COVID-19) is a severe threat tohumankind because of rapid contagion, increased morbidity, andmortality, and extreme patient suffering.

One consequence of this pandemic is the need to wear a facial coveringto prevent CoV inspiration or to reduce CoV expiration from an infectedperson. But the wearing of masks, especially the tight-fitting onescalled N95 filtering facepiece respirators (N95-FFR), for periods oflonger than one hour are uncomfortable, especially in hot weather. Thetechnology here addresses the problem of mask and respirator discomfort[NIOSH, 2015. Workplace solutions: preparedness through daily practice:the myths of respiratory protection in healthcare. Krah J et al.,Cincinnati, Ohio].

Pathogenesis of Airborne Transmission and the Need for Masks

Coronaviruses (CoV), a subfamily of Coronavirida, are RNA viruses withgenomes of ˜30 Kb. Of the seven known human coronaviruses, four causemild symptoms such as nasal congestion and cough. The dangerous CoV areβ-CoV, namely, MERS, SARS-1, and SARS-2 (CoV-2), which attack the lowerrespiratory tract (LRT) and cause pneumonia.

“Tropism” describes how a virus selectively contacts its host cell viaan attachment receptor. The point of contact is an essential determinantof disease infectivity, virulence, and pathogenicity. The viral tropismfor 3-CoV is attachment to a protein cell membrane receptor called theACE-2 receptor (for an angiotensin-converting enzyme, type 2) [Hoffmann,M. et al., 2020. “The Novel Coronavirus 2019 (2019-NCoV) Uses theSARS-Coronavirus Receptor ACE2 and the Cellular Protease TMPRSS2 forEntry into Target Cells.” BioRxiv, 2020.01.31.929042]. The virusrecognizes ACE-2R on host cells via its S-spike proteins, and it is theanatomical distribution of ACE-2R that decides the sites of infectionand pathogenesis. After infection and propagation, the next generationof virions emerge from the host cell, like a bunch of frog eggs, andcause havoc.

Anatomically, the boundary of the upper respiratory tract (URT) and LRTis at the glottis. The conducting airways of the LRT have on its luminalsurface epithelium and goblet cells and sub-divide for 20 to 22generations to become the gas exchange surface know as the alveolus (pl.alveoli). The alveoli contain Type 1(AT1) and Type 2(AT2) pneumocytes.AT1 cells are flat and allow gas exchange between alveolar air and thepulmonary capillaries. AT2 cells, which are ˜2.5% of the total cells inthe alveolus, are cuboidal and synthesize surfactant, a material thatprevents the alveoli from collapsing. The host molecule and bindingpartner of the β-CoV for the virus are angiotensin-converting enzyme-2(ACE-2R). The S-spike protein of CoV binds to ACE-2R, fuses with thecell membrane, and enters the AT2.

Once the virus enters the AT2 cell, it utilizes the cell's machinery andreplicates itself. At some point, the AT2 disintegrates and releases thevirions. The outpourings of new virions are abundant. Immune cells, suchas macrophages and neutrophils, recognize the foreign virions andrelease cytokines to begin an attack on the invaders. The resulting“cytokine storm” causes alveolar destruction, loss of oxygen exchange,tissue damage, pneumonia, and fever. In highly susceptible individuals,these events can be explosive, and the infected person can die withinfive days. Pneumonia accounts for the high morbidity and mortality ratesseen with the β-CoV.

The CoV has a long journey through 20+ generations of airways to reachthe ACE-2 receptor on AT2 cells. A direct route for the virions to reachtarget is via droplets that remain suspended in air and gets inhaleddeeply to reach the alveoli. These droplet diameters are <10 microns(μm). The virus itself has a diameter of about 0.5 μm, but it exists inthe air as an aerosol (liquid plus solid), so the bioaerosol has agreater diameter. The human eye can detect particles of about 30 μm, butthe small inhalable CoV-2 particles are not visible. The CoV-2 can alsomove downward from the conducting airways in the alveolar lining fluids(ALF) to the alveoli, but the mucociliary clearance mechanism brushesthe ALF upwards towards the larynx. From swab tests for CoV-2, we knowthat CoV-2 is present in the nasal cavity and on the throat. Incubationof the virus in the URT may generate significant quantities of CoV-2 tooverwhelm mucociliary clearance and gain access to the AT2.

The mode of transmission of CoV-2, close-range aerosol spread, dictatesthe imperative use of masks, or facial covering, as a barrier to preventinfection.

Personal Protection Equipment, Principal Types of Masks

CoV-2 is a contagious pathogen, and healthcare workers (HCW) havesuccumbed to this virus. The factors that contribute to increased risk,such as long hours of close contact in the ICU, require wearing awell-fitted N95-facial filtration respirator (FFR) type of mask togetherwith a protective gown (FIG. 1). Alternative procedures for protectionfrom infection include physical (social) distancing, cough etiquette,and handwashing but are not effective for close patient contact (<3feet) [Nicas et al., Relative Contributions of four exposure pathways toinfluenza infection risk. Risk Anal., 2009]. “Personal protectiveequipment” (PPE), which includes respirators, face masks, gloves,³ eyeprotection, face shields, gowns, and head and shoe coverings, arephysical interventions to prevent the entry of CoV into the body. Forthe airways, respirators are designed to purify or filter inhaled air.N95 (95% efficiency) filtering-facepiece respirators (N95-FFR) aremanufactured from electrostatic filter material such as polypropylene,which yield high collection efficiency due to electrostatic attractionof charge particles and low breathing resistance. By contrast, facemasks (FM), also called surgical masks, are loose-fitting coverings madefrom fabric and designed to protect the patient from secretions from thenose and mouth of the HCW. Face masks are not designed to protect thewearer from exposure to respiratory hazards but do have some value asbarriers to penetration of pathogens into the wearer. For healthcarepersonnel in close contact (defined <3 feet) with symptomatic patients,PPE is N95 (95% efficiency) filtering-facepiece respirators (N95-FFR),safety eyeglasses, and disposable clothing. These methods of protection[National Academies Press. Reusability of facemasks during an influenzapandemic: facing the flu. Chpt. 2. Characteristics of respirators andmedical masks. NAP.edu/10766. Washington, D.C. 20001] follow thepostulated pathways for viral transmission. Reduced exposure via therespiratory route of exposure has the highest priority.

Face masks (FM), also called surgical or medical masks, are designed toprevent the wearer's release of large droplets into the environment. FMdo not protect much from small particulates (i.e., droplet nuclei) thatcan harbor pathogens But, it protects HCW from large-droplet splashes orsprays of bodily fluids from patients. FM are typically disposable andloose-fitting, do not form a tight seal to the face, or capture smallparticles efficiently. By contrast, N95-FFR filter at least 95% of awide size-range of particles and must be N95-FFR custom-fitted for thewearer. N95-FFR are named where “N” stands for not oil resistant and tobe used for oil-free atmospheres. The N95-FFR is for the hospital roomswhere there is a high risk of pathogen transmission. In the past 40years, the dominant N95-FFR is a filtration piece made of non-wovenmaterials such as polypropylene, The N95-FFR removes ˜95% of particles,down to a particle size of 0.3μ. N95-FFR, because of its tight fit tothe face, may cause skin irritation, a buildup of facial heat, and anincrease in airflow resistance. Prolonged and continual use, e.g., >4 hris not well-tolerated and may lead to a failure of compliance oradherence of use.

A third type of respirator is elastomeric respirators (ER), which aretight-fitting respirators where the facepieces are made of synthetic ornatural rubber material and can be repeatedly used, cleaned,disinfected, stored, and re-used. These are alternatives to disposableFM and filtering facepiece respirators (FFR), such as N95 FFR. The ERprovides equivalent protection to N95-FFR, and some types of ER offerhigher assigned protection factors than N95 FFR. The ER has replaceablefilter cartridges or flexible, disc, or pancake-style filters. All ERequipped with the proper air-purification filters, cartridges, orcanisters have utility in personal protection. Elastomerics may alsohave sealing surfaces and adjustable straps that accommodate a betterfit. The limits of ER is the expense and re-usability.

Li et al. [2005. Effects of wearing N95 and surgical facemasks on heartrate, thermal stress, and subjective sensations. Int Arch Occup EnvironHealth; 78:501-9] compared FM and N95-FFR in healthy subjects working atcontrolled temperature and humidity. After ˜80+ min, subjects wearingthe N95-FFR started to complain of excess heat and humidity andincreased breathing resistance. There was an increase in heart rate andblood pressure, indicative of stress and sympathetic nervous systemactivation. The N95-FFR subjects felt unfit, fatigued, and complained ofoverall discomfort. This evidence of heat stress from N95-FFR masks ismanifest in clinical environments, e.g., in the Emergency Departments ofhospitals treating CoV infections. Farquharson reported that working12-h shifts while wearing an N95-FFR mask was a serious challenge totheir staff [2003. “Responding to the Severe Acute Respiratory Syndrome(SARS) Outbreak: Lessons Learned in a Toronto Emergency Department.” J.Emergency Nursing 29 (3): 222-28].

Effectiveness of Masks and PPE

Tracht et al. [2010. “Mathematical Modeling of the Effectiveness ofFacemasks in Reducing the Spread of Novel Influenza a (H1N1).” PLoS ONE5 (2)] modeled the effectiveness of FM and N95-FFR in reducing thespread of influenza for 2009 H1N1. He estimated that when masks orrespirators were absent, the infected population was 75%; if 10% woreFM, the percentage infected was 73%; if 50% wore FM, the infectedpopulation was 69%. By contrast, if 10% wore N95-FRR, the infected was55%; and if 50% wore N95-FFR, they would drop to 0.1%. These results arespeculative and not proven by an experiment.

In a more direct study of effectiveness, MacIntyre et al. [A randomizedclinical trial of three options for N95 respirators and medical masksfor clinical health workers. Amer. J Critical Respiratory Care. 187,960-966, 2013] compared the use of facial coverings in 14 hospitals. HCWwere put into groups of FM, continuous use of N95-FFR, or targeted useof N95-FFR (while doing high-risk procedures or barrier nursing).Outcomes were clinical respiratory illness (CRI). The results showed CRIwas highest in the FM group (17%), followed by the targeted N95 arm(11.8%), and lowest in the continuous N95 arm (7.2%) (P<0.05). MacIntyreconcluded that N95-FFR was more efficacious against CRI thanintermittent use of N95-FFR or medical masks.

The use of personal protective equipment and history of high-riskpatient care activities among SARS-exposed nurses [Loeb et al. 2004.“SARS among Critical Care Nurses, Toronto.” Emerging Infectious Diseases10 (2): 251-55] found that three (13%) of 23 nurses who consistentlywore a mask (either surgical or N95-FFR) acquired SARS compared to 5(56%) of 9 nurses who did not consistently wear a mask. The relativerisk (RR) for infection was 0.22 and statistically significant, p=0.06.

Sensory Events on the Facial Areas Covered by a Mask and Mask Discomfort

A standard N95-FFR mask covers the skin over the bridge of the nose, thecheeks, the mouth, and the chin. The triangular area enclosed by thenasolabial folds (smile lines), alar crease, the nostrils, vermilion,and the lower lip, is sensitive to heat. The tip of the nose and theskin of the nares sense air temperature and humidity. In the respiratoryepithelium, within the nasal cavity, receptive fields are present,especially around Kiesselbach's plexus (Little's area). The cranialnerves transmit somatosensory and thermosensory signals from the face,nasal cavity, and mouth. These signals are highly integrated intotemperature regulation, as exemplified by the observation that coolingof the face is two to five times more effective at suppressing sweatingand thermal discomfort than cooling an equivalent skin area elsewhere onthe body [Cotter JD, Taylor NA. (2005). The distribution of cutaneoussudomotor and alliesthesial thermosensitivity in mildly heat-stressedhumans: an open-loop approach. J Physiol; 565: 335-45].

Reasons for Face Mask Discomfort

Roberge et al. [2012. “Protective Facemask Impact on HumanThermoregulation: An Overview.” Annals of Occupational Hygiene 56 (1):102-12] reviewed the postulated reasons for FFR and FM discomfort.Factors considered were respiratory heat exchange mechanisms, thepathways of heat gain and loss from the nose and mouth, the thermoloadof protective facemasks, facial skin temperature, dead space heat andhumidity, and psychophysiological heat responses. No conclusions werereached on the best strategy to reduce discomfort. Suggested mitigationmeasures were refrigeration of masks, miniature-battery powered fans,altering the geometry of masks, and exhalation valves to reduce heataccumulation. Only exhalation valves have reached the market, but thebenefits appear limited. The valves add to the respirator's expense andare banned if the wearer's exhalation is likely to infect others. Thepharmacological treatment of the mask discomfort has not beencontemplated.

In summary, the inhalation of a β-CoV is an urgent worldwide threat tohealth. The interposition of a barrier, in the form of a mask (FM orN-95-FFR), has a protective function of filtration. In a pandemic, thebenefits of wearing a facial cover by HCW are overwhelming andself-evident. The use of masks is now ubiquitous, but sensory discomfortfrom a mask is a hindrance to continued and adherent use.

BRIEF SUMMARY OF THE INVENTION

The CoV pandemic of 2019+ has increased the need for efficient use ofpersonal protection equipment (PPE), as the number of sick exponentiallyincreases. A necessary part of protection is a mask filter barrier toprevent the entry of the virion into the nose and mouth of the wearer. Amask barrier reduces respiratory transmission, the most dangerous routeof viral infection. Inhaled virions reach their host cell in the alveolivia the conducting airways. Preventing the entry of inhaled particles isan established method of protection. In the hospital, especially inintensive care units, health care workers (HCW) must wear tight-fittingmasks for long hours, sometimes with impermeable gowns. The resultingsensory discomfort leads to non-adherence to the use of masks and anincreased risk of infection. A method to reduce sensory discomfort fromwearing a mask has value.

Wearing a facial covering is an artificial and unnatural event, but nowfrequent because of the pandemic. A face mask (FM) or an N95-FFR(filtering facepiece respirator) covers a defined region of the face,which includes the cheeks, and the nasal and perioral areas. But afacial covering is a barrier to air exchange. Heat and water vaporaccumulates behind the mask and acts on thermosensors primarily onfacial skin. What are the precise anatomical sites and mechanisms ofmask discomfort?

In the laboratory and field studies, the descriptions of sensorydiscomfort of masks give a list of signs and symptoms. Firstly, subjectswearing fitted N95 respirators complain of increased resistance toairflow when breathing. Then, there are complaints of heat buildupbehind the mask, especially in a hot environment. Finally, there arecomplaints of loss of concentration, perceptual disorientation,headache, lightheadedness, and impeded communication [Rebmann et al.2013. “Physiologic and Other Effects and Compliance with Long-TermRespirator Use among Medical Intensive Care Unit Nurses.” AmericanJournal of Infection Control 41 (12): 1218-23]. Somatic complaints areof an increased heart rate, shortness of breath, skin irritation, andfeelings of nausea. These complaints increase with the duration ofcontinuous use, beginning in one laboratory study after 80 min. Mostwearers find work-shift durations of 4 hr, 8 hr, and 12 hr to beunpleasant. In an infected area, however, protocols for the removal of amask are strict, laborious, and tedious, so masks are mandatory.

To ascertain the sites and mechanisms of discomfort, we experimented. Acooling agent, called a TRPM8 agonist, was topically applied todifferent sites of the face of volunteers to determine the site thatbest counteracts the discomfort caused by wearing an N95 FFR for 1 to1.5 hr. A TRPM8 agonist skin gel containing 1.5% (15 mg/mL) of DIPA-1-7was applied with a forefinger at 0.03 to 0.05 mL to discrete sitesnormally covered by a mask: namely, 1. Alar crease. 2. External nares.3. Lateral cheek. 4. Nasolabial folds. 5. Philtrum. 6. Vermilion, and 7.Chin, as shown in FIG. 6 Surprisingly and unexpectedly, all sites gavecoolness and cold sensations at various intensities, but only gelapplication on the external nares gave robust and effective relief offace mask discomfort. Thus, for coolness and cold, the rank order of thesites was alar crease >external nares philtrum >vermilion nasolabialfolds folds >chin >>lateral cheek. For example, the application of thegel to the nasolabial folds or lips did not relieve discomfort, whereasapplication to the skin of the external nares (nostrils) was instantlyeffective. Further discussion with the test subjects illuminated themechanism of drug action.

The test subjects stated that if the nose felt cold, air movement witheach breath also felt fresh and cold, and the resistance to breathingdisappeared. If the skin felt cold on the nasolabial folds or the chin,this refreshed breathing was not present, and the discomfort inbreathing remained. This result also provides a hypothesis for why masksare uncomfortable. Constant breathing of static warm air causesdiscomfort, not heat or humidity on the skin. Refreshed breathing is adynamic event. With a mask, the subject feels suffocated because the airdoes not move. With cooling, there is a relief of discomfort. Earlierstudies showed that dynamic and not static neuronal discharges into thebrain account for the sensation of cooling in humans. This refreshingcooling is a dynamic event that requires the discharge of cooling TRPM8nerve fibers. DIPA-1-7 gel on the nostrils enhances this neuronal event.

To further characterize this discovery, a method for selective drugdelivery to the skin of the external nares was improvised. The hollow ofthe volar palm forms a receptacle that holds about 0.5 to 1 mL ofliquid. An ideal method to deliver a drug to the external nares is toplace a solution into the hollow and immerse the nose tip into thehollow for ˜5 sec. This technique compares molecular potency andduration. For example, to construct dose-response data, a TRPM8 agonistsolution was stored at a fixed volume in a disposable reservoir unit (3mL polyethylene bottle) at 0.25 to 4 mg/mL solution in saline. Next, 0.8mL aliquot is dispensed onto the hollow of the palm. Then the anteriornose is immersed into the hollow at a ˜45° angle for 5 sec, as shown inFIG. 7. The tip of the nose fits snugly into the palmar cavity andprecisely delivers the test solution to the skin of the nostrils.Inhalation of the solution into the nasal cavity itself is optional butnot necessary. Alternatively, use a gel in the hollow of the palm.

Surprisingly, this method of contacting the skin of the external nareswith a TRPM8 agonist yielded reliable dose-response data, as shown inFIG. 8. In practice, a single local application of DIPA-1-7 to 3 at 2mg/mL in saline is sufficient to overcome mask discomfort for at least 4hr and can be repeated with equal effect for another 4-hr period. Bycomparison, localized wiping of the DIPA-1-7 to other facial areas, suchas the upper lip, philtrum, lips, and chin, were less effective forreducing mask discomfort.

The mechanism of action of DIPA-1-9 for the relief of mask discomfortcan be described in non-technical language. The skin of the nose tip,especially around the nostrils, has sensors for temperature in the coolrange of less than 25° C. These “thermistors” or TRPM8 nerve endings areactivated by cold, such as on a cold day or on the ski slope, whereinthe subject has a “runny nose.” The sensors also convey the pleasure ofbreathing cool air by the seaside or on a breezy day. The sensors shutoff behind a face mask when airflow is impeded or occluded, and heataccumulates above 25° C. There is no air movement. In the natural statethe sensors respond best to the rate of temperature change, not statictemperature. Applying a TRPM8 receptor agonist, such as DIPA-1-9 to theskin of the nostrils, restores and enhances the thermosensitivity tocoolness. The air-conditioning system re-adjusts, and the subject feelsbetter with a sense of cooler airflow when breathing. Less attention ispaid to the annoyance of the facial covering.

An essential part of this discovery is the recognition that the skinaround the opening of the nostrils is highly sensitive to thermalstimuli and influences airflow comfort and discomfort. The second partof the discovery is the identification of selective TRPM8 agonistmolecules that can be precisely delivered to the nostril site to relievemask discomfort. The prior art does not record this method of selectivetopical delivery of a cooling agent to the external nares skin for thetreatment of mask discomfort.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1. is an illustration of filtering face masks that covers the noseand mouth of a wearer. Left: a facial mask, also called a surgical maskor medical mask. Right: an N95 facepiece filtering respirator.

FIG. 2. shows the cooling sensations evoked by topical wiping ofdifferent concentrations of DIPA-1-8 onto the skin above the zygomaticprocess. The cooling activity can be measured as the intensity/durationarea-under-curve (AUC) or as the time for half-maximal effect(T_(−1/2)), using software of the GraphPad Prism package. The graphshows the dose-response curve for the compound DIPA-1-8 applied at 0.5,1, and 2% (5, 10, and 20 mg/mL dissolved in distilled water).

FIG. 3. shows the cooling sensations evoked by the topical wiping ofdifferent compounds onto the skin above the zygomatic process. Thecooling activity is expressed as the integrated intensity/durationarea-under-curve (AUC), using the software of the GraphPad Prismpackage. Test concentration was 2% (20 mg/mL in distilled water). Thediisopropyl analogs are DIPA 3,3-X and isopropyl, sec-butyl analogs areMixed 3,4-X. The “X” refers to the number of carbons on the third alkylgroup. The Mixed analogs are much less active on the cheekbone skin thanthe corresponding diisopropyl analogs.

FIG. 4. is a graph of fluorescence response (Δ ratio 340/380) in TRPM8transfected cells as a function of the logarithm of the concentration ofthe test compound, expressed in μM, for DIPA-1-7 (black circle), 3,4-7(open squares), or 3,4-6 (open triangles). The assays were conducted byAndersson et al. of King's College, London, UK, using his methodsdescribed in “Modulation of the cold-activated channel TRPM8 bylysophospholipids and polyunsaturated fatty acids. Journal Neuroscience27 (12): 3347-3355, 2007.

FIG. 5. is an illustration of the human face showing the test sites forapplying a gel containing 1.5% (15 mg/mL) of DIPA-1-7. The sites areidentified by the numbered circles: 1. Alar crease. 2. External nares.3. Lateral cheek. 4. Nasolabial folds. 5. Philtrum. 6. Vermilion. 7.Chin. A 0.03 to 0.5 mL aliquot of a commercial gel containing 1.5% ofDIPA1-7 (Intrinsic IB, Dong Wha Pharmaceuticals, Seoul, Republic ofKorea) was applied with a forefinger to the designated site.

FIG. 6. shows the flux of DIPA-1-7 and DIPA-1-9 through excised hairlessmouse skin in vitro. Test chemicals dissolved in a gel were placed in anincubation well for 8 hr and the permeated amount of the chemicalmeasured by a high-pressure liquid chromatograph equipped with arefractive detector. These tests were conducted by Prof Choi of ChosunUniversity, Korea. The flux of 1-7 was ˜5×greater than 1-9. Standardenhancers with polyhydric alcohols, such as a propylene glycol-oleylalcohol mixture (50:50) or Lauroglycol 90, designed to increasepermeation added to the 1-7 gel decreased the rate of permeation by˜50%, indicating the importance of water solubility for permeation.

FIG. 7. is an illustration of a bioassay procedure of a cooling agentapplied to the external nares. A 0.8 mL volume of the test substance isplaced in the hollow of the palm (gray area). The tip of the nose (at˜45° angle) was immersed into the center of the hollow for 5 sec. Thecontact points on the nostrils are starred.

FIG. 8. is an illustration of the cooling effects of DIPA-1-9 applied tothe external nares using the hollow of the palm assay. Test results werefor 0.25, 0.5, 1.0, 2.0 and 4.0 mg/mL of DIPA-1-9 dissolved in salineand placed at 1 mL in the hollow of the palm for immersion of theexternal nares for ˜5 sec.

DETAILED DESCRIPTION OF THE INVENTION

In the current CoV pandemic, HCW such as doctors and nurses use anN95-FFR (filtering facepiece respirator), which is fitted to the wearerand reduces risks of infection. The general public mainly wears a facemask (FM), which is loose-fitting and protects less against the virus.The facial covering in both types of masks leads to discomfort aftercontinuous use, especially when the environment is hot. The exact causesof mask discomfort are not known. The discovery here suggests that acooling agent applied to the skin above the nostrils effectivelyprevents and reduces mask discomfort.

Sensory Discomfort of Masks and Respirators

A facial covering is an artificial and unnatural event that is a barrierfor air and heat exchange. The mask covers the skin over the triangularsurface area delineated by the nose's bridge, the nasolabial folds(smile lines), and the lower lip and parts of the chin. Heat and watervapor accumulates behind the mask and acts on thermosensors on the skinand nasal cavity. Airflow is confined and static. The enclosed surfacesof the skin of the nostrils, the alar crease, and the vermilion aresensitive to heat. Receptors that sense air temperature and humidity arealso densely present in the anterior nasal vestibule and the respiratoryepithelium of Kiesselbach's plexus (Little's area). The sensory signalsfrom the facial skin and nasal structures are transmitted by the facialnerve and the trigeminal nerve to the brain and integrated intotemperature perception and regulation.

The dynamic sensitivity of the facial area and nasal cavity to heat ishigher than the sensitivity of the torso's skin and centrally integratedfor perception. Cooling of the face is two to five times more effectiveat suppressing sweating and thermal discomfort than cooling anequivalent skin area elsewhere on the body (Cotter and Taylor, videsupra). The greater sensitivity of the facial skin to coolness isself-evident when one uses an air-conditioner. The cool air is moreeffective when blown over the face than over the body.

When the use of the mask is prolonged, there is heat accumulation behindthe mask, and the upper lip skin temperature increases by 1 to 2° C. Thesubjects complain of increased resistance to airflow when breathing,heat behind the mask, difficulty in communication to others, and skinirritation. There is also a loss of concentration, perceptualdisorientation, and increased heart rate. These signs and symptoms arecommon in subjects exposed to environmental heat stress, but in maskusers, the heat stress is only on the face and not the rest of the body.

Respirators and masks protect if HCW adheres to use. In an 8-hr workshift, 59% of the workforce discontinued wear, citing communicationdifficulties (visual, auditory, or vocal), heat, pressure or pain, anddizziness or difficulty concentrating. Median tolerance times rangedfrom 4.1 to 7.7 hours. Wearers of disposable models often complained offacial heat and pressure, while users of reusable models often reportedcommunication problems. A cup-shaped N95-FFR with an exhalation valvehad a better median tolerance time (7.7 hours) than a similar modelwithout a valve (5.8 hours). In a survey of users, only 24% reportedthat their N95-FFR respirator was comfortable most of the time oralways, but only 6% said that they would tolerate wearing it for an 8-hrshift. The problems identified were discomfort, difficult breathing,heat, and low tolerability for an extended time.

In a controlled environment study of 10 nurses, the compliance rate totwo-12 hr shifts wearing an N95 respirator was 78%. There were, however,subjective symptoms of perceived shortness of breath, complaints ofheadache, lightheadedness, hindered communication, and feelings ofnausea.

MacIntyre et al. (vide supra) showed that facial coverings protect HCWagainst chronic respiratory illnesses. Masks also protected nursesduring a SARS outbreak in Toronto. Thus, increased adherence orcompliance to wearing a mask may be a life-saving event (see Loeb et al.vide supra).

Cryosimulator Unit

A cryosimulator unit is a unit for the delivery of a TRPM8 agonist toits defined receptive field [Wei, E. T. 2020. “Improving Brain Power byApplying a Cool TRPM8 Receptor Agonist to the Eyelid Margin.” MedicalHypotheses 142 (April): 109747.https://doi.org/10.1016/j.mehy.2020.109747]. The delivery unit may be atopical gel, a unit dispenser with a fixed volume solution, a rinse, aswab, or a wipe.

A water-soluble TRPM8 agonist such as DIPA-1-9, applied topically,instantly cools the nasal area, gives a sense of free-breathing, andrelieves heat discomfort. The best cryosimulator unit for maskdiscomfort is a gel or a solution applied via the hollow of the palm tothe external nares. The cryosimulator unit here acts topically like amini air conditioner for the nasal skin. In addition to the skin,another source of dynamic thermal information comes from the nerveendings of the transitional epithelia inside the nasal vestibule. Therespiratory epithelium of the nasal cavity is not the primary target.Nasal sprays are not preferred because the hospital environment maycreate airborne infectious particles. The skin above the nares is idealbecause the air move over these thermosensors. Other facial skin, suchas those innervated by the facial or infraorbital nerve, may contributeinformation on the status of coolness/cold in the external environment,but the response dynamics are less sensitive.

The receptor coding for and transducing cool/cold signals follows adiscrete “cable” line coded by the receptor called TRPM8 [Knowlton etal. 2013. A sensory-labeled line for cold: TRPM8-expressing sensoryneurons define the cellular basis for cold, cold pain, andcooling-mediated analgesia. J. Neurosci. 33, 2837-48]. The sensoryneurons for TRPM8 do not overlap with the neurons coded for heat, suchas the TRPV1 receptor. Thus, the mechanism of neuronal interactions isindirect. TRPM8 signals suppress heat signals centrally in the brain,not in the periphery.

The detailed molecular characteristics and physiology of TRPM8 areknown. Amino acid residues important for binding and function areidentified, and the TRPM8 structure has been clarified by cryo-electronmicroscopy to a resolution of ˜4 Ångstroms [Pertusa et al., 2018.Critical role of the pore domain in the cold response of TRPM8 channelsidentified by ortholog functional comparison. J. Biol. Chem. (2018) 293:12454-1247; Yin, Y. et al., 2019. Structural basis of cooling agent andlipid sensing by the cold-activated TRPM8 channel. Science. 2019 Mar. 1;363]. When the temperature is ≤25° C., TRPM8 pores open up, and entry ofcations across nerve endings triggers action potentials to the brain,activating cool/cold signals from the specific receptive field. Chemicalcooling agents such as menthol or icilin facilitate the opening of TRPM8pore. TRPM8 nerve endings discretely aggregate in the body [Dhaka, etal., 2008. Visualizing Cold Spots: TRPM8-Expressing Sensory Neurons andTheir Projections. J. Neurosci. 28, 566-575]. In rodents, there is ahigh density of innervation in the whisker pad and the skin.

Mechanism of Action, Receptor Target, Selection of Active Ingredient

Relief of mask discomfort requires the selection of the right moleculeas the correct receptor agonist. The target location must be defined,and the formulation and concentration prepared in a delivery system togive sufficient duration of action. The time for delivery is optimizedto when cooling is needed.

An agonist is a chemical activator of biological events [Reuse, J. J.,1948. Comparison of various histamines antagonists, Brit. J. Pharmacol.3: 174-180]. A TRPM8 agonist acts on a binding site of TRPM8 tofacilitate the opening of the receptor pore. After entry of cations intothe pore, action potentials are generated and transmitted into thecentral nervous system. The threshold for feeling cool goes down. Forthe selection of an active ingredient, there are four major classes ofTRPM8 agonists: a) the monoterpenes as represented by 1-menthol, b)icilin, c) p-menthane carboxamides and related cyclohexane derivatives,and d) 1-dialkylphosphorylalkanes. Menthol does not work on the nasalskin. It irritates and causes rhinorrhea. Icilin is practicallyinsoluble in any solvent, so delivery is problematic. The p-menthanecarboxamides and esters need a solvent system before delivery, but gelsor creams are possible. By experiment, ideal agents identified here arewater-soluble 1-dialkylphosphorylalkanes called DIPA-1-8 [Cryosim-2,1-diisopropylphosphoryloctane, CAS Registry No. 2959-63-9], and DIPA-1-9[Cryosim-3, 1-diisopropylphosphorylnonane, CAS Registry No.1507344-37-7]. These compound at active concentrations of 1 to 3 mg/mLdissolve in water or saline and has the desired cooling actions. Yang etal. [2017. A novel TRPM8 agonist relieves dry eye discomfort BMCOphthalmology (2017) 17:101, 1-15. doi:10.1186/s12886-017-0495-2]described the pharmacology of DIPA-1-9. DIPA-1-7[Cryosim-1,1-diisopropylphosphorylheptane, CAS Registry No.1487170-15-9] is also effective but less preferred because it produces astinging cold and causes rhinorrhea and has a shorter duration ofaction.

Delivery System

An essential part of hypothesis testing is to use a gel, solution, orwipe to deliver a cool TRPM8 agonist to the receptors on the skin of theexternal nares. A familiar form of drug delivery is to use a gel appliedwith a finger to the nostril skin. Alternatively, a formulation for thepalm hollow, with instructions to rub a subject's nose tip into thehollow, works well. These methods are preferable to wipes because thedelivery is exactly over the nares, but the methods require carefulinstructions to the user. The topical route avoids the likelihood ofsystemic side effects. For a gel, an adequate volume is ˜0.05 to 0.2 mLand for a liquid, an aliquot of ˜0.6 to 1.0 mL. For a solution, instructsubject to incline face at ˜45° and place nose tip into palm hollow for˜5 sec, as show in FIG. 7. For a rinse, an adequate volume is 5 to 20 mLand the concentration of the DIPA compound is 0.05 to 0.25 mg/mL.

For each molecule, activity is a sum of penetration, distribution,localization, and intrinsic actions at the receptor. The discovery ofthe best compounds for the skin of the nares is by iterative experiment.The preferred embodiments for mask discomfort are1-[Dialkyl-phosphinoyl]-alkanes [(O═)PR1R2R3] wherein each of R1, R2, iseither isopropyl or sec-butyl and R3 is a linear alkyl group of 6 to 9carbons, and wherein the embodiments have 15 or 16 carbons. Suchcompounds are DIPA-1-8 and DIPA-1-9, and, for the sec-butyl analogs, 2-6and 2-7.

The ideal agent must act locally, and the intensity of the sensationshould not cause “icy cold,” coldness in the chest, or systemic chills.One preferred embodiment is DIPA-1-9 because it is water-soluble doesnot over-activate the cold receptors in the nose tip skin. To show thepreparation works requires ease of formulation and use, rapid onset ofaction, for example, within 1 to 3 min after application, absence ofirritation at the site of application, absence of systemic effects, andsufficient duration of action to prevent mask discomfort. Further proofrequires a randomized, double-blind, and placebo-controlled trial.

TERMINOLOGY AND ANATOMICAL DESCRIPTIONS

Cryosimulator unit (CSU) A cryosimulator unit is a unit for the deliveryof a TRPM8 agonist to its defined receptive field. The delivery unit maybe in the form of a bolus of water, a wipe, a dropper with a reservoir,a unit dispenser, a rinse, a swab, or a topical gel. In this discovery,the preferred CSU is a gel or a 1 mL volume of liquid (e.g., 1 mL ofDIPA-1-9 at 2 mg/mL in saline) placed in the cup of the volar palm, andapplied to the nostril of the mask wearer with instructions to contactthe liquid content.Intrinsic® IB Spot Serum from Donghwa Pharmaceutical Co., Ltd., Seoul,Republic of Korea, which contains 1.5% wt/vol of DIPA-1-7 as its maincomponent, was used as the “active” test gel. The placebo gel consistedof the same excipients present in Intrinsic IB, but without theDIPA-1-7. Neither the patients nor the clinician investigator was madeaware of the component of the prescription. The patients applied a smallamount with the fingertip as needed, to the topical area.DIPA compounds DIPA is the abbreviation for1-[Diisopropyl-phosphinoyl]-alkane or 1-diisopropylphosphorylalkane. Anumber describes the third alkyl group in the molecule: hence, 4, 5, 6,7, 8, 9, and 10 correspond to the butyl, pentyl, hexyl, heptyl, octyl,nonyl, and decyl side chain, respectively. These alkanes are linear or“normal [n]” in configuration, with the phosphinoyl group attached tothe primary, or “1-” position, of the carbon chain in the thirdsidechain. These compounds are also known as trialkylphosphine oxides oras 1-dialkylphosphorylalkanes.Dynamic Cooling. Small myelinated (Aδ) and unmyelinated fibers (Cfibers) increase afferent firing rate when skin temperature decreases,for example, between 35° C. and 15° C. The neuronal signals that detectheat abstraction transmit to the central nervous system for perceptionof coolness and cold. Raising temperature from 35° C. and 40° C.increases firing rates in C fibers to signal warmth [Hutchinson et al.Quantitative analysis of orofacial thermoreceptive neurons in thesuperficial medullary dorsal horn of the rat. J. Neurophysiol. 77,3252-66, 1997]. The receptive mechanisms and “cable lines” for cool/coldand warm are separate and distinct, but reciprocally inhibit each otherin the brain and perhaps also in the periphery. The sensory receptorsare modality-specific and do not respond to mechanical stimulation. Atthe molecular level, the target binding sites for cooling agents are onTRP ion channel receptors that depolarize in response to a drop intemperature. Heat abstraction decreases the threshold for discharge ofthe receptor, and the facilitated depolarization initiates the axonalresponses that create the neuronal signal.

The central response of these neurons has been recorded and studied fromrat superficial medullar dorsal horn that responds to innocuous thermalstimulation of the rat's face and tongue [Hutchinson et al., 1997]. Stepchanges of −Δ5° C. stimulated cells with both static firing rates andcells that had mainly dynamic properties [Davies et al. Sensoryprocessing in a thermal afferent pathway. J. Neurophysiol. 53:429-434,1985]. Similar studies in cats and humans showed that stepdecreases in temperatures (dynamic changes), as low as Δ0.5° C./second,were readily detectable by neurons and by psychophysical measurements[Davies et al. Facial sensitivity to rates of temperature change:neurophysiological and psychophysical evidence from cats and humans. J.Physiol. 344: 161-175, 1983].

A study of the spike patterns of neuronal discharge (impulses/second)showed that dynamic, and not static firing responses to a change intemperature were the most potent stimuli for generating coolness/coldsensations. That is, the brain “sees”−Δ ° C./t and not absolute ° C.Thus, a cooling agent that simulates −Δ ° C./t on nerve discharge evokes“dynamic cooling.”

Face Masks (FM) Face masks, also called surgical or medical masks, areloose-fitting coverings of the nose and mouth, designed to protect thepatient from secretions from the nose or mouth of the physician, nurse,or other healthcare professional. Face masks are also used by the wearerto reduce exposure to respiratory hazards.N-95-Face Filtration Respirators (N-95-FFR) Protective respirators workby filtering the air inspired by the wearer. N95-FFR are cleared by theFood and Drug Administration (FDA) as medical devices. Effectiverespirators must fit tightly to the face.Facial mask discomfort and heat stress Thermal comfort is a technicalterm used by air-conditioning engineers to define “a state of mind inhumans that expresses satisfaction with the surrounding environment.”Maintaining thermal comfort for occupants of buildings or otherenclosures is one of the essential goals of architects and designengineers. For most people, the room temperature for thermal comfort is20 to 25° C. Careful studies have documented that work performance andproductivity (output/input) drop by 2% for every increment of +1° C.above 25° C. up to 33° C. At office temperatures of 28-30° C. (82-86°F.), there is sweating and complaints of headache, drowsiness, anddullness, difficulty in concentrating, and physical discomfort and lossof work performance [Tanabe et al., 2007. Indoor Air Quality andProductivity. REHVA Journal 44(2) 26-31]. An ambient temperature above25° C. is thus a form of heat stress. Symptoms and signs of general heatdiscomfort are similar to mask discomfort.Healthcare Workers (HCW) or Healthcare Personnel broadly encompass allworkers in direct patient care and support services who are employed byprivate and public healthcare offices and facilities as well as thoseworking in home healthcare and emergency medical services, includingthose who are self-employed.Heat Discomfort of Wearing Masks There are three main types of maskdiscomfort. One is an increased effort in breathing, i.e., there is asense of resistance to airflow caused by fabric in the mask. A secondsensation is that of accumulated heat behind the mask with sensations ofsuffocation and impaired ventilation. The third type of symptom isdisturbed perception, a lack of concentration, and an inability toperform routine tasks efficiently.Hollow of the Palm Assay. The cavity in the center of the volar palmformed by the thenar and hypothenar muscle contraction convenientlyforms a receptacle for the nares. Palmists call it “Plain of Mars,” andthe centerline “Fate Line.” To assay, load hollow with ˜0.05 to 1 mL andimmerse nares into hollow for ˜5 sec.Nares. The nares (singular, naris), or nostrils, are the pair ofopenings immediately below the tip of the nose and are the entrances tothe respiratory tract. The nasal passages serve as a conduit forinspired and expired air. When these passages have increased resistanceto flow, are congested, or obstructed, subjects perceive it asuncomfortable. The nostril sill is defined as the soft tissue bulgebetween 4 borders; medially the foot plate of the columella, laterallythe ala, cephalically the vestibule, and caudally the upper lip. Thesill consists of fibro fatty tissue composed of a mixture of dermalcollagen and glands. The skin above the nares are sensitive to heat andcold.Nasal patency—is the subjective sensation of openness and smooth airflowin the nasal passages when breathing. Loss of patency may be reported as“nasal stuffiness” or “nasal blockage” and causes discomfort, and thesubject may use mouth breathing, which is undesirable because itdesiccates the airway surfaces. Nasal congestion implies excess fluidsin the nasal passages. The term “nasal obstruction” means there is astructural hindrance to airflow. Rhinitis, the inflammation of themembranes in the nasal cavity, is most often associated with nasalcongestion and nasal obstruction. In the “empty nose syndrome,” thereare severe breathing discomforts, including loss of the sense ofpatency, but the symptoms can occur without rhinitis or physicalevidence of change in airflow or gas exchange [Sozansky, J.Pathophysiology of empty nose syndrome. Laryngoscope 125, 70-74 (2015)].Note here that relief of mask discomfort is an action on the skin andnot on nasal patency.The nasal afferents for detecting temperature are on branches of thetrigeminal nerve. Receptors that mediate detection of coolness is thecation channel called TRPM8. Keh et al. [The menthol and cold sensationreceptor TRPM8 in normal human nasal mucosa and rhinitis. Rhinology 49,453-7 (2011)] have detected TRPM8 immunoreactivity in the human nasalmucosa, closely associated with nerve fibers and blood vessels.Breathing cool air, for example, at the seaside, enhances the sense offresh airflow in the nose. In the laboratory, subjects report a greatersense of nasal patency with lower nasal septum temperatures [Willatt etal. The role of the temperature of the nasal lining in the sensation ofnasal patency. Clin. Otolaryngol. Allied Sci. 21, 519-523 (1996)]. Peakmucosal heat loss in a critical region of the anterior nose is a keycorrelate of the sense of nasal patency [Zhao, K. et al. Regional peakmucosal cooling predicts the perception of nasal patency. Laryngoscope124, 589-595 (2014)]. The benefit of cooling the skin of the nose tip isa new concept.Breathing cool air increases the sense of nasal patency. Inspired airtemperature at the septum kept at 25 to 35° C., gives a greater sense ofpatency at the lower temperature [Willatt et al., vide supra]. However,it is also well-known that cold and frigid air evokes a “runny nose” orrhinorrhea, an event mediated by cholinergic nerves on serous glands ofthe nasal epithelium [Ostberg et al. Cold air induced rhinorrhea andhigh-dose ipratropium Arch. Otolaryngol. Head and Neck Surg. 113,160-162 (1996)]. This condition has also been called a “skier's nose”and is quite common. Thus, the cooling of the nasal skin must avoidrhinorrhea, which is not desirable for a person wearing a mask. In aprevious study, the preferred embodiments, DIPA-1-8 and DIPA-1-9, testedat a total dose of ˜50 μg cooled the nasal cavity but not the nostrilskin. These DIPA compounds require a higher dose on the facial skin formask discomfort. DIPA-1-8 and DIPA-1-9 were selected for the currentindication of face mask discomfort because they were long-acting.Philtrum Assay. The test site of drug application is the skin above theupper lip (above the vermilion border of the lips), on the philtrum(midline groove), lateral to the philtrum until the nasolabial folds,and sometimes, but not deliberately, on the lower nostrils (subnasale).This part of the face is densely innervated with “cold spots” secondonly to the surfaces of the eyeball and anogenitalia. At this locus,cool and cold sensations in the skin may be experienced and rated foronset and intensity. Test substances dissolved in ointment (e.g.,Aquaphor®, which is 41% petrolatum, and the rest mineral oil, ceresin,and lanolin alcohol) or (R)-1,2-propanediol and singly applied onto theskin surface using a cotton-tipped stick. The sensation intensity ratingis 0, 1, 2, or 3 with 0 as no change; 1 as slight coolness, cold, ortingling; 2 as clear-cut signal of coolness, cold, or tingling; and 3 asrobust cooling or cold. The intervals for recording sensations are 5 to10 minutes till two consecutive zeroes.Receptive field of a sensory neuron is the region in space in which astimulus modifies the firing of the neuron. The receptive field is thespatial distribution of the nerve endings of the neuron. For theepithelium, the nerve endings interdigitate with the cell layers at theepithelium's basal layer. A receptive field, as small as an mm², whenactivated by the appropriate stimulus, e.g., nociceptive or pruritic,can dominate the brain's and mind's attention. Witness what happens whena sharp pin or sting comes into contact with skin or when a dog ispre-occupied with a flea bite.Skin, Keratinized Epithelia, and Permeation Barriers. There are fourbasic types of animal tissues: connective tissue, muscle tissue, nervoustissue, and epithelial tissue. Epithelia line ducts, cavities, andsurfaces of organs throughout the body. When the layer is one cellthick, it is called simple epithelium. If there are two or more layersof cells, it is called stratified epithelium. Stratified epithelium iscomposed mainly of squamous (flattened) cells and some cuboidal cells.Historically, stratified epithelia divide into two broad categories:keratinized stratified epithelia, and non-keratinized stratifiedepithelia. Keratinized epithelium, such as the epidermis of the skin,has an exterior layer of dead cells [stratum corneum] composed ofkeratin proteins that are tough and water-impermeable. Keratin alsocovers the filiform papillae of the tongue. By contrast,non-keratinizing stratified epithelia do not contain a significantkeratin layer and are present principally on: the lining of the nasalcavity; portions of the oral cavity such as the inner lips; thepharyngeal surface; the esophageal surface; the lining of therespiratory tree; and the anogenital surface. The term “cornifiedepithelia” has traditionally been reserved for keratin-covered tissuessuch as nails, hair, and hooves [Bragulla and Homberger [Structure andfunctions of keratin proteins in simple, stratified, keratinized andcornified epithelia. Journal of Anatomy, 214: 516-59, 2009].

The skin of the nares is keratinized epithelia. As it merges into thenasal vestibule and nasal cavity, it transitions to the non-keratinizingrespiratory epithelium. Transitional epithelia in the body are generallydensely innervated with sensory nerve endings [e.g., the margins of theeyelids, the inner border of the lips, and the margins of theanogenitalia]. A layer of keratin is a barrier for drug access toneuronal receptive fields embedded in tissues underneath the keratin.Biologists now recognize keratin as a family of proteins that formintegral filaments in the cytoskeleton of many cells. Hence, the term“non-keratinized stratified epithelia” is no longer accurate and mayeventually become obsolete. The preferred term is “non-keratinizingstratified epithelia,” implying that these cells do not form an externalkeratin layer. In the context of this application, the skin above thenostrils is “keratinizing stratified epithelia.” This skin becomestransitional epithelia as it merges with the nasal vestibule and thenbecomes respiratory epithelia, which is non-keratinizing. Respiratoryepithelium is a single cell layer know as pseudostratified epithelia.

TRP channels The transient receptor potential (TRP) family of cationchannels are peripheral detectors of nociceptive and painful stimuli.Many of these receptors are located on the nerve membranes of sensoryneurons and respond to chemical irritants and changes in localtemperature by activating nerve action potentials. The brain perceivesthese signals and react. The TRP receptors transduce sensoryinformation, and this transduction and reflex system protects theorganism from external irritants.Thermosensory Mechanisms on the Skin One of the essential advances inphysiology, in the past 20 years, is the discovery that physical changessuch as heat, cold, and pressure are sensed and transduced by integralmembrane proteins on the cell surface. Typically, in an ambientenvironment of 18 to 25° C., the skin temperature remains constant at˜34° C. The thermoreceptor is in the extracellular fluid of the skin.The receptors coding for and transducing temperature belong to the TRPfamily of cation channel proteins. The receptive fields of thethermosensors for mask discomfort are in the trigeminal nerve. Thesereceptors sample static and dynamic changes in local temperature. TRPM8nerve endings for cooling discretely aggregate in the body [Dhaka etal., vide supra]. The high density of nerves on the whisker pads ofrodents is equivalent to the perioral areas of humans. Other sites arethe nasal mucosa and lips.

Study 1 DIPA Compounds

The present discovery relates to certain compounds (the DIPA compoundsdescribed herein), which, when delivered onto the skin, selectively andpotently evoke sensations of “dynamic cool” when applied to the facialskin. These compounds have applications in the treatment of face maskdiscomfort.

The structures of the preferred embodiments are shown below. Thewater-soluble compounds [e.g., 1-di-isopropyl-phosphinoyl-heptane]potently [<5 mg per dose] and rapidly produce on skin robust and intensecooling sensations. This type of drug action is unusual and has not beenpreviously recognized as achievable on keratinized surfaces and has ledto new applications, as described herein.

The DIPA compounds of the present discovery are achiral and are examplesof 1-di-alkyl-phosphinoyl-alkanes [(O═)PR₁R₂R₃] wherein each of R₁, R₂,and R₃ is an alkyl group, and in particular where R₁ and R₂ areisopropyl, and R₃ is a linear alkyl group of 5 to 9 carbons, and whichhave the following general formula of Formula 1:

Chemical Synthesis

The DIPA compounds were prepared by the following general method: 100 mL(23.7 g, ˜200 mmol) of isopropylmagnesium chloride (orsec-butylmagnesium chloride in the case of the di-sec-butyl derivatives)were obtained from Acros, as a 25% solution in tetrahydrofuran (THF) andplaced under nitrogen in a 500 mL flask (with a stir bar). Diethylphosphite solution in THF (from Aldrich, D99234; 8.25 g, 60.6 mmol in 50mL) was added drop-wise. After approximately 30 minutes, the reactionmixture warmed up to boiling. The reaction mixture was stirred for anextra 30 minutes, followed by drop-wise addition of the appropriaten-alkyl iodide solution in THF (from TCI; 60 mmol in 20 mL). Thereactive mixture was then stirred overnight at room temperature. Thereaction mixture was diluted with water, transferred to a separatoryfunnel, acidified with acetic acid (˜10 mL), and extracted twice withether. The ether layer was washed with water and evaporated (RotaVapBuchi, bath temperature 40° C.). The light brown oil was distilled undera high vacuum. The final products, verified by mass as determined bymass spectrometry, were transparent liquids that were colorless.Professional chemists conducted synthesis at Phoenix Pharmaceuticals,Inc. (Burlingame, Calif.), Uetikon Laboratories (Lahr, Germany), andDong Wha Pharmaceuticals (Seoul, Korea).Table 1 compounds were prepared by this general synthetic method andused for comparisons.

TABLE 1 DIPA compounds Formula/ Code Chemical Name Weight ChemicalStructure DIPA- 1-5 1-di-isopropyl- phosphinoyl-pentane C₁₁H₂₅OP 204.32

DIPA- 1-6 1-di-isopropyl- phosphinoyl-hexane C₁₂H₂₇OP 218.32

DIPA- 1-7 1-di-isopropyl- phosphinoyl-heptane C₁₃H₂₉OP 232.34

DIPA- 1-8 1-di-isopropyl- phosphinoyl-octane C₁₄H₃₁OP 246.37

DIPA- 1-9 1-di-isopropyl- phosphinoyl-nonane C₁₅H₃₃OP 260.40

TABLE 2 Chemical structures of test compounds. Chemical Code NameChemical Structure 2-4 1-di(sec- butyl)- phosphinoyl- Butane

2-5 1-di(sec- butyl)- phosphinoyl- Petane

2-6 1-di(sec- butyl)- phosphinoyl- Hexane

2-7 1-di(sec- butyl)- phosphinoyl- Heptane

2-8 1-di(sec- butyl)- phosphinoyl- Octane

3-1 1-di(iso- butyl)- phosphinoyl- Petane

3-2 1-di(sec- butyl)- phosphinoyl- 3- methyl-butane

TABLE 3 Chemical structures of “Mixed” test compounds. 3,4-61-isopropyl- sec-butyl- phosphinoyl- hexane

3,4-7 1-isopropyl- sec-butyl- phosphinoyl- heptane

3,4-8 1-isopropyl- sec-butyl- phosphinoyl- octane

3,4-9 1-isopropyl- sec-butyl- phosphinoyl- nonane

The 3,4-X series are “mixed” isopropyl-sec-butyl compounds (Table 3).These were synthesized by Dr. Jae Kyun Lim of Dong Wha Pharmaceuticals,using the method described below.Briefly, as illustrated in the following scheme, triethyl phosphite (A)was reacted with sec-butyl magnesium bromide (B) and then hydrolyzedwith dilute hydrochloric acid to give the mono-alkyl compound (C). Theproduct (C) was then reacted isopropyl magnesium bromide (D) to give thedi-alkyl compound (E), which was then reacted with a suitable alkyliodide (F) to give the target trialkyl phosphine (G).

General Observations of Unusual Properties

DIPA compounds are colorless liquids with a density less than water. Thepreferred embodiments DIPA-1-7, DIPA-1-8, and DIPA-1-9 exert an icysensation that can modulate skin dysesthesia caused, for example, byvarious dermatitis (e.g., atopic or urticarial) and on mucous membranes(esp. DIPA-1-8 and DIPA-1-9). Similar structures were described byRowsell and Spring U.S. Pat. No. 4,070,496 (1978) ˜40+ years ago buthave remained dormant in the scientific literature. The '496 structures(see table) ALL have their “head” (phosphine oxide group) covered bylarger, more lipophilic groups. The applicant noted that '496 did notinclude the di-isopropyl analogs. The applicant synthesized theseanalogs (which are achiral, by contrast to the structures of '496, whichare >95% chiral). The applicant found that, by minimizing the two alkylside chains to di-isopropyl, the “head” of the prototypical molecule nowis more polar (hydrophilic) and more miscible in the polar environmentof water. This increased water-solubility is striking (Table 4). Thewater solubility of the DIPA if at least 10×more than the di-sec-butylor the mixed isopropyl-sec-butyl analogs. The DIPA analogs are nowmobile in the extracellular fluids and permeate between cells to accessnerve endings in the stratum basale.

TABLE 4 Water solubility (mg/ml) of 1-dialkylphosphorylalkanes (R₁R₂R₃P= O). No. Carbons 13 14 15 16 R₁, R₂ R₃ R₃ R₃ R₃ di-sec-butyl- pentane22 hexane 8 heptane <3 octane <3 isopropyl-sec-butyl- hexane 25 heptane20 octane <3 nonane <3 di-isopropyl- heptane >300 octane >300nonane >300 decane <3

Facial application of DIPA compounds as an aqueous solution at 1-10mg/mL or a 1% hydrogel, is non-irritating. For certain analogs,contacting the skin with a solution at a concentration of 1 to 10 mg/mLproduces a sensation of “dynamic cool,” which occurs within one minuteafter application. DIPA-1-7, especially, has intense dynamic cooling.

Compositions

In U.S. Ser. No. 15/530,976 filed Mar. 31, 2017 and U.S. Ser. No.16/350,559 filed Nov. 30, 2018, of which this application is acontinuation in part, DIPA compound compositions and methods ofpreparing DIPA compound compositions were described.

In one embodiment, the composition comprises the DIPA compound at aconcentration of 0.05 to 2.0% wt/vol. In one embodiment, the compositionis a liquid or semi-liquid composition (gel, lotion, cream, orointment), and comprises the DIPA compound at a concentration of 0.5 to20 mg/mL. In one embodiment, the composition is liquid rinse andcomprises the DIPA compound at a concentration of 0.05 to 0.25 mg/mL inwater or isotonic saline. In one embodiment, the composition is liquidand comprises the DIPA compound at a concentration of 1 to 5 mg/mL inwater or isotonic saline. In one embodiment, the composition is liquidand comprises the DIPA compound at a concentration of 5 to 10 mg/mL. Inone embodiment, the composition is liquid and comprises the DIPAcompound at a concentration of 10 to 20 mg/mL.

The composition may be provided with suitable packaging and in asuitable container. For example, the composition may be as a rinse,swab, wipe, pad, or towelette (e.g., suitably sealed in a wrap) carryinga DIPA compound or a composition comprising a DIPA compound. In anotherexample, the composition is provided as a solution in a single unitdispenser, e.g., in a volume of 1 mL per dispenser, for example, asmanufactured by Unicep Corporation (1702 Industrial Drive, Sandpoint,Id., USA)

In one embodiment (e.g., of use in methods of therapy, of use in themanufacture of medicaments, of methods of treatment), the treatment isthe treatment of sensory discomfort caused by wearing a face mask. Theterm “sensory discomfort”, as used herein, relates to unpleasantsensations from the facial surface such as suffocation, dyspnea, orheat. The term implies activation of nociceptors located on sensorynerve endings of the body. Nociceptors are stimulated, for example, byhigh temperatures or air stasis. A DIPA compound, such as DIPA-1-8 orDIPA-1-9 that decreases sensory discomfort, is termed ananti-nociceptive agent. In one embodiment, the treatment conveys a senseof refreshment to the skin in a human.

For a liquid vehicle or semi-liquid vehicle, a preferred deliveredvolume is 0.05 to 2.0 mL. Such a volume, delivered for example as asolution, gel, lotion, or a wipe, does not cause much residual liquid atthe delivery site, as is absorbed onto the skin. For a liquid vehicle, apreferred concentration of the DIPA compound is in the range of 0.5 to30 mg/mL. For the facial skin, a preferred concentration is 1 to 5mg/mL. For the zygomatic and infraorbital skin, a preferredconcentration is 5 to 10 mg/mL. For the forehead skin and scalp, apreferred concentration is 10 to 30 mg/mL. A preferred amount of theDIPA compound delivered at the site of the application is 0.1 to 5 mg;for example, 0.1 to 5 mg.

Wiping the DIPA compound on the target skin can be done withpre-medicated wipes, which are well-known in personal care products.Usually, these wipes are packaged as a single-use sealed unit or in amulti-unit dispenser. For single units, suitable wrapper materials arerelatively vapor impermeable, prevent drying out of the wipe, and form a“peelable” seal. Examples of suitable wipe materials for practicing thisdiscovery include polyamide (20% Nylon)-polyester, rayon (70%)-polyester(30%) formed fabric, polypropylene nonwoven, polyethylene terephthalate(PET), polyester polypropylene blends, cotton, viscose, rayon, ormicrofibers (synthetic fibers that measure less than one denier or onedecitex).

Alternatively, a solution containing a DIPA compound is in a reservoirbottle with individual applicators, or as a pre-packaged individualunit. For example, Puritan 803-PCL applicators are ideal cotton-tippedapplicators attached to a 3-inch (˜7.5 cm) polystyrene rod for deliveryof a DIPA compound onto the periorbital skin. Examples of applicatorsindividually packaged are the SwabDose™ from Unicep Corporation (1702Industrial Drive, Sandpoint, Id., USA), and the Pro-Swabs from AmericanEmpire Manufacturing (3828 Hawthorne Court, Waukegan, Ill., USA). Eachapplicator tip is saturated by dipping the absorbent material of the tip(e.g., 40 to 100 mg of cotton) in 0.1 to 1.5 mL of an aqueous solutionof a DIPA compound and packaged in an individual container.

For application to the face, instructions are for the individual togently apply the cream, lotion, gel, or wet wipe onto the subject'sfacial skin. Instructions may include teaching the individual to repeatapplication or “topping up” to ensure that sufficient compositionreaches the target. For nostrils, a gel can be applied directly.Alternatively, a solution goes onto the hollow of the palm and thesubject immerses the nose tip into the hollow for ˜5 sec. The immersionmethod is unusual but effective.

Selection of Active Ingredient

An active pharmaceutical ingredient (API) for delivery to the face'skeratinized skin should be stable, non-toxic, and long-acting. The APIshould potently activate the mechanisms that result in the relief offace mask discomfort. The API should dissolve and evenly disperse in acomposition so that during manufacture, the formulation maintains aconstant concentration. The final product should meet the standards ofcleanliness and sterility. For formulation, the API can be a liquid atstandard conditions of temperature and pressure (STP). It shoulddissolve in aqueous solutions at neutral pH and isotonicity. The finalproduct is sterile by using purified reagents and micropore filters,heating, or irradiation. Standard solvents such as water or isotonicsaline, stabilizing agents, and preservatives, may be added to optimizethe formulations, but the essential ingredients should be preferablysoluble in aqueous media such as purified water or a standarddermatological solvent.

For a given individual, the perceived sensation is a function of theparticular cooling agent, the dose, the vehicle used to carry thecooling agent, the method of topical delivery, and the nature of thetarget surfaces. The applicant has screened many candidates on thefacial skin (Wei. Sensory/cooling agents for skin discomfort. JournalSkin Barrier Research 14: 5-12, 2012). The studies here identifypreferred DIPA compounds with properties of an ideal agent for therelief of face mask discomfort.

Study 1 General Parameters of Sensory Effects of Compounds on FacialSkin

Compound tested on the skin produces a characteristic pattern ofsensations. The quality of cooling sensations evoked, their descriptors,and their proposed mechanism of action, are summarised in Table 5. Foreach compound, there is usually only one or two categories ofoverlapping sensations. For example, icilin is exclusively cool, withvery little “cold.” DIPA-1-6 and DIPA-1-7 produce a robust “dynamiccool” that can elicit “stinging” sensations at higher concentrations.DIPA-1-8 and DIPA-1-9 are potent cold-producing agents.

TABLE 5 Descriptor and proposed mechanisms of DIPA compounds on skin.Proposed Mechanisms on Type of Sensation Descriptor Sensory NeuronsInactive No effect — Cool, steady and pleasant Cool Balanced stimulationof static and dynamic Cold, constant Cold Higher stimulation of staticDynamic cooling, robust Dynamic Higher stimulation cool/cold, strongrefreshing cool of dynamic Stinging cold, sometimes Icy cold Stimulationof with irritation dynamic and static, and also nociceptive sites

After the offset of the cooling/cold action, some compounds have a“reservoir effect.” Experimentally, this is measured 1 hour after anoffset by placing a hot and then a cold towel over the site ofapplication and determining if the onset of cooling/cold returns for atleast 30 minutes. If this occurs, then there is a definite “reservoireffect.” The “reservoir effect” can also be provoked with air movement,but the conditions for air movement are difficult to standardize. The“reservoir effect” of DIPA-compounds in the skin is most likely due toresidual drug deposited in the skin, which reactivates to stimulatedynamic/static sensory neurons.

In the studies described herein, the sensation of coolness/cold is ratedas 0, 1, 2, or 3 with 0 as no change; 1 as mild coolness; 2 as clear-cutsignal of coolness; and 3 as robust cooling. The sensations are recordedat intervals of 5 to 15 minutes until two consecutive zeroes.

The onset of drug action is taken as the time to reach 2 units ofcoolness intensity. The duration of sensory action is defined as theoffset time minus the onset time. An inactive compound is defined as onethat does not exceed 2 units of cooling for 5 minutes or more afterapplication. The offset endpoint is sometimes unstable for compoundsthat act for two or more hours, because the coolness/cold sensation mayfluctuate due to environmental variables such as sunlight, ventilation,activity, and the “reservoir effect.”

Tables 6 show the effects of test compounds on zygomatic facial skin.Test compounds were applied 20 mg/mL, in distilled water to the skin ofthe zygoma using cotton gauze (0.4 g, rectangular, 50 mm×60 mm; fromCS-being, Daisan Cotton, Japan). The onset and duration of the sensoryeffect was measured with a stopwatch. The degree of “dynamic cool” wasgraded from 0 to +++, with intermediate steps of + and ++.

TABLE 6 Sensory effects after application to zygomatic and foreheadskin. Carbon Onset Sensory Duration Reservoir Code R₃ atoms (min)Quality Anti-Fatigue (hr) Effect DIPA-1-5 5 11 ~1 dynamic 0 0.5 NoDIPA-1-6 6 12 ~1 dynamic ++ 1.3 Yes DIPA-1-7 7 13 ~1 dynamic-icy +++ 3.2Yes DIPA-1-8 8 14 ~1 cold-icy ++ 4.0 Yes DIPA-1-9 9 15 ~2 cool 0 2.0 No2-4 4 12 ~1 cool 0 0.3 No 2-5 5 13 ~1 cool 0 1.1 Yes 2-6 6 14 ~2 cold +1.5 Yes 2-7 7 15 ~2 cold + 2.4 Yes 2-8 8 16 5 cold 0 5.6 YesEach of 3-1 and 3-2 was tested and found to be inactive on periorbital,and zygomatic/forehead skin.

For further comparisons, the newly synthesized “Mixed”1-isopropyl-sec-butyl-phosphorylalkanes (3,4-6, 3,4-7, 3,4-8, and 3,4-9)were tested on zygomatic skin (FIG. 3). The test procedures weremodified because of the limited quantities of these analogs. To deliverthe solution to the skin, an 80%-polyester-20%-viscose rayon wipe wascut into squares (7×8 cm, 0.45 g each) and a precise volume (2.5 mL) oftest solution added to the wipe using a dropper bottle. An average 74±2μL volume containing the test ingredient was wiped onto the receptivefields of the nerves on the zygomatic process (cheekbone). As before,the sensory effects of cool/cold were recorded at 5 and 10 minintervals. Quarter and half-point scores are allowed. The scoringstopped when two consecutive zeroes occurred in a 10 min interval. Twoto three volunteers was used for each compound. Results from the testingof DIPA-1-8 at three concentrations are shown in FIG. 2.

FIG. 2. shows the cooling sensations evoked by topical wiping ofdifferent concentrations of DIPA-1-8 onto the skin above the zygomaticprocess. The cooling activity can be measured as the intensity/durationarea-under-curve (AUC) or as time for half maximal effect (T_(−1/2)),using software of the GraphPad Prism package. The graph shows the AUCdose-response curve for the compound DIPA-1-8 applied at 0.5, 1 and 2%(5, 10, and 20 mg/mL dissolved in distilled water).

A comparison of the DIPA diisopropyl analogs (3,3-X) versus the mixedpropyl-sec-butyl analogs (3,4-X) are shown in FIG. 3. Statisticalsignificant differences (P<0.01) are seen between 3,3-x and theasymmetrical chiral 3,4-x analogs. The 3,4-8 and 3,4-9 formed amilky/small oil droplet emulsion at 20 mg/mL. Notably, DIPA-1-7selectively produced the unusual sensation of “dynamic cool.” From thedata shown above, it was apparent that DIPA-1-7 evoked “dynamic cool” onboth periorbital and zygomatic/forehead surface. Another compound withsimilar properties was DIPA-1-8, but this compound is was more cold/icycold, although it had the desirable property of a longer duration ofaction on the zygomatic/forehead surface. The long duration of action ofDIPA-1-8 and DIPA-1-9 on the skin adds value as an agent for thetreatment of mask discomfort. As shown in the case studies describedbelow, a single application of DIPA-1-8 or DIPA-1-9 is sufficient tocounteract mask discomfort for at least three to four hours. Theexceptional value of DIPA-1-9 is the comfortable cooling it provides andits long duration of action after application onto the nostril skin andthe absence of any stinging. Thus, it has an essential therapeutic nichefor the relief of face mask discomfort. These results for the particularattributes of DIPA-1-7, DIPA-1-8, and DIPA-1-9 are unexpected,surprising, and has practical applications for counteracting face maskdiscomfort.

Study 2 Receptor Agonist Activity of Compounds on TRPM8

The in vitro effects of the first set of test compounds (Table 1 and 2)were evaluated on cloned hTRPM8 channel (encoded by the human TRPM8gene, expressed in CHO cells) using a Fluo-8 calcium kit and aFluorescence Imaging Plate Reader (FLIPR^(TETRA)™) instrument (conductedby ChanTest Corporation (14656 Neo Parkway, Cleveland, Ohio 44128, USA).

Test compounds and positive control solutions were diluted stockHEPES-buffered physiological saline (HBPS) solutions. The test andcontrol formulations were loaded in polypropylene or glass-lined384-well plates, and placed into the FLIPR instrument (Molecular DevicesCorporation, Union City, Calif., USA). Tests were 4 or 8 concentrationswith n=4 replicates per determination, and the positive controlreference compound was L-menthol, a known TRPM8 agonist. The test cellswere Chinese Hamster Ovary (CHO) cells stably transfected with humanTRPM8 cDNAs.

For FLIPR^(TETRA)™ assay, cells plated in 384-well black wall, flatclear-bottom microtiter plates (Type: BD Biocoat Poly-D-Lysine MultiwellCell Culture Plate) were approximately 30,000 cells per well. Cells wereincubated at 37° C. overnight to reach a near confluent monolayerappropriate for use in a fluorescence assay. The test procedure was toremove the growth media and to add 40 μL of HBPS containing Fluo-8 for30 minutes at 37° C. 10 μL of the test compound, vehicle, or controlsolutions in HBPS were added to each well and read for 4 minutes.Concentration-response data were analyzed via the FLIPR Control softwarethat is supplied with the FLIPR System (MDS-AT) and fitted to a Hillequation of the following form:

${RESPONSE} = {{Base} + \frac{{Max} - {Base}}{1 + \left( \frac{xhalf}{x} \right)^{rate}}}$

where: “Base” is the response at low concentrations of a test compound;“Max” is the maximum response at high concentrations; “xhalf” is theEC₅₀, the concentration of test compound producing half-maximalactivation; and “rate” is the Hill coefficient. Nonlinear least-squaresfits were assumed based on a simple one-to-one binding model. The 95%Confidence Interval was from the GraphPad Prism 6 software. The resultssummary is in Table 8.

The EC₅₀ of the more potent compounds (DIPA-1-7, DIPA-1-8, DIPA-1-9,2-5, 2-6, 2-7, 2-8) fell within a narrow range with overlapping 95%Confidence Intervals. The potency of DIPA-1-7, DIPA-1-8, and DIPA-1-9are similar and significantly higher than the potencies of DIPA-1-5 andDIPA-1-6. By contrast, the structural modifications of comparativecompounds 3-1 and 3-2 resulted in a significant loss of bioactivity.Further studies examined the specificity of the test compounds on TRPV1channels (human TRPV1 gene expressed in HEK293 cells) and TRPA1 channels(human TRPA1 gene expressed in CHO cells). The test cells were ChineseHamster Ovary (CHO) cells or HumanE embyronic Kidney (HEK) 293 cellstransfected with human TRPV1 or TRPA1 cDNAs.

TABLE 8 EC₅₀ and relative potency of compounds on TRPM8.. 95% ConfidenceRelative Code EC₅₀ μM Interval Potency Menthol 3.8 2.5 to 5.6 1.0DIPA-1-5 5.6 4.4 to 7.2 0.7 DIPA-1-6 2.4 1.5 to 4.0 1.6 DIPA-1-7 0.7 0.5to 1.0 5.4 DIPA-1-8 0.7 0.5 to 1.0 5.4 DIPA-1-9 0.9 0.4 to 2.5 4.0 2-414.5 7 to 29 0.3 2-5 1.7 1.0 to 2.9 2.2 2-6 0.8 0.5 to 1.3 4.7 2-7 1.10.6 to 2.3 3.4 2-8 1.3 0.7 to 2.3 2.9 3-1 24 8 to 76 0.2 3-2 4.2 1.6 to10.8 0.9

Of the 12 compounds tested, all showed full efficacy on the TRPM8receptor, i.e., at higher tested concentrations, there was ˜100%stimulation of calcium entry, and the data fitted a sigmoidaldose-response curve. The results for the “di-isopropyl” compounds ofthis invention are illustrated in FIG. 3.

Selectivity tests were conducted on Human Embyronic Kidney (HEK) 293cells transfected with human TRPV1 or TRPA1 cDNAs. The positive controlreference compound was capsaicin (a known TRPV1 agonist) or mustard oil(a known TRPA1 agonist). DIPA-1-7 and DIPA-1-8 did not exhibit anyagonist on antagonist activity on TRPA1 channels at maximum testedconcentrations of 100 μM. DIPA-1-7 had a weak TRPV1 agonist activity,but this was not dose-dependent.

Further tests were conducted on “mixed”isopropyl-sec-butylphosphorylhexane and heptane analogs. The data werecollected by Andersson et al. of King's College, London, UK, using hismethods described in “Modulation of the cold-activated channel TRPM8 bylysophospholipids and polyunsaturated fatty acids. Journal Neuroscience27 (12): 3347-3355, 2007. Here, the cellular entry of thecalcium-sensitive dye Fura-2 was used to study the effect of the testcompounds on TRPM8 expressed in Chinese hamster ovary cells. Cells,grown in culture, were seeded at an approximate density of 30,000cells/well overnight and loaded for ˜1 hr with 2 M Fura-2 (MolecularProbes, Leiden, The Netherlands), and then placed on glass coverslips.Test solutions were added with a micropipette positioned close to thecells. Emission intensity from cells was measured for 90 sec, at every 4or 5 sec, using excitation wavelengths of 340 and 380 nm and an emissionof 520 nm. Fluorescence emission intensity ratios at 340 nm/380 nmexcitation (R, in individual cells) were recorded with a FlexStation andthe ImageMaster suite of software (PTI, South Brunswick, N.J.). Sampleswere tested in triplicate at each concentration and the averaged valuesanalyzed by nonlinear regression using a sigmoidal function fit of thepoints to obtain an estimated EC50 (median effective concentration)(GraphPad Prism software, La Jolla, Calif.).

The potency of three analogs for activation of TRPM8 (cooling receptor)in transfected cells is shown in FIG. 4. The units (Δ ratio) on theordinate measures entry of fluorescent calcium probes into transfectedcells. The 3,3-7 (DIPA-1-7) is substantially more potent (˜10× and ˜5×)than 3,4-6 and 3,4-7. Note that 3,4-6 and 3,4-7 species do not reach thesame degree maximal efficacy on activation of the receptor, even atsupra-maximal concentrations. From these results, it appears that theEC₅₀ values do not give information on the quality of the heatabstraction sensation, the duration of action, or the accessibility ofthe molecule to tissue targets. The identification of selective agentsrequires bioassays that more directly address these questions.

FIG. 4. is a graph of fluorescence response (Δ ratio 340/380) in TRPM8transfected cells as a function of the logarithm of the concentration ofthe test compound, expressed in μM, for DIPA-1-7 (black circle), 3,4-7(open squares), or 3,4-6 (open triangles). The assays were conducted byAndersson et al. of King's College, London, UK, using his methodsdescribed in “Modulation of the cold-activated channel TRPM8 bylysophospholipids and polyunsaturated fatty acids. Journal Neuroscience27 (12): 3347-3355, 2007.

Study 3 Physical Chemistry, Water Solubility and Penetration ofCompounds to Target

The receptor targets on nerve endings embed and interdigitate in theepithelial cell layers. The epidermis is only ˜1 mm thick, but dead celllayers (stratum corneum) of denatured proteins impede access of theagonist molecule to the nerve endings. The heel of the feet is thethickest barrier, 86 cell layers for the heel, and followed by the palm,then the back of the hand. If you put an ice cube on the heel, you feela bit of cold: but you jump when you put it on the sole of the feet,which has fewer layers. Unless the skin of the thick surfaces isstructurally damaged, e.g., by inflammation, applying a cooling agentdoes not work, because the molecules do not access the nerve endings.For other surfaces, the genital skin (glans of the penis and vulva) andthe eyelids are the thinnest, with 4 to 8 cell layers. The extremities,arms and legs, and the trunk (back) have thick surfaces. The scalp isintermediate. The face varies: the lateral cheek is relativelyinsensitive, but areas around the orbit and nasolabial folds are thinand sensitive. I estimate that the cell layer thickness for the cheek is15 to 18 cell layers, and 7 to 15 cell layers for the nostrils,philtrum, alar crease, nasolabial folds, vermillion, and chin. Thesedifferences are critical for drug action! For cooling to relieve maskdiscomfort, choose the molecule carefully to get the desired effects:avoid too much stimulation and exert gentle cooling.

The applicant's preferred embodiments of DIPA-1-7, DIPA-1-8, andDIPA-1-9, wherein two of the alkyl groups (e.g., R₂ and R₃) are bothisopropyl, have high water solubility and skin penetration. Increasingwater solubility to increase bioactivity is counterintuitive, as instandard drug design, the desire is to increase lipid solubility toenhance transdermal drug permeation. Usually, formulation experts try tobreak down the stratum corneum with enhancers, and chemists try toincrease lipid solubility of the molecule (see, e.g., M. Prausnitz etal. Skin barrier and transdermal drug delivery. Chpt. 124, MedicalTherapy, 2012). The strategy used here was, however, met with clinicalsuccess.

To further study the skin permeation of DIPA compounds, we tested theflux of DIPA-1-7 and DIPA-1-9 through excised hairless mouse skin invitro (FIG. 5).

FIG. 5. shows the flux of DIPA-1-7 and DIPA-1-9 through excised hairlessmouse skin in vitro. Test chemicals dissolved in a gel were incubatedfor 8 hr and the permeated amount of the chemical measured by ahigh-pressure liquid chromatograph equipped with a refractive detector.These tests were conducted by Prof Choi of Chosun University, Korea. Theflux of 1-7 was ˜5×greater than 1-9. Standard enhancers with polyhydricalcohols, such as a propylene glycol-oleyl alcohol mixture (50:50) orLauroglycol 90, designed to increase permeation added to the 1-7 geldecreased the rate of permeation by ˜50%, indicating the importance ofwater solubility for permeation.

Standard enhancers with polyhydric alcohols, such as a propyleneglycol-oleyl alcohol mixture (50:50) or Lauroglycol 90, designed toincrease permeation added to the 1-7 gel decreased the rate ofpermeation by ˜50%, indicating the importance of water solubility forpermeation. In studies on the abdominal skin of anesthetized rats, a50:50 propylene glycol-DIPA-1-7 mixture was inactive when tested on theskin of animals, with shaking as an endpoint, whereas the pure DIPA-1-7was very active. Thus, standard solvents or enhancers of dermatologicalmolecules impede rather than facilitate passage of the DIPA through theskin barriers. The mobility of the DIPA molecules in an aqueousenvironment through a skin barrier is unusual and surprising. The“unmasking” of the polar “head” by one or more carbon (e.g., methyl)groups, increases water solubility and permeability. The symmetrical(achiral) arms (the isopropyl groups) may also enable efficient swimmingof the DIPA through the pores of the stratum corneum and into theextracellular fluid to reach the TRPM8 receptors in the stratum basale

Study 4 Identification of Target Sites for Face Mask Dismcomfort:Anatomical Localization

A standard N95-FFR mask covers the skin over the bridge of the nose, thecheeks, the mouth, and the chin. The triangular area enclosed by thenasolabial folds (smile lines), alar crease, the nostrils, vermilion,and the lower lip, is sensitive to heat. The tip of the nose and theskin of the nares sense air temperature and humidity. In the respiratoryepithelium, within the nasal cavity, receptive fields are present,especially around Kiesselbach's plexus (Little's area, see Zhao et al.,vide supra). The cranial nerves transmit somatosensory and thermosensorysignals from the face, nasal cavity, and mouth. The facial signals arehighly integrated into temperature regulation, as exemplified by theobservation that cooling of the face is two to five times more effectiveat suppressing sweating and thermal discomfort than cooling anequivalent skin area elsewhere on the body (Cotter and Taylor, videsupra).

The head is known to be a site where cooling helps relieve heatdiscomfort. Nakamura et al. [2013. “Relative Importance of DifferentSurface Regions for Thermal Comfort in Humans.” European J. Appl.Physiol. 113: 63-76] exposed eleven male subjects to mild heat.Subjects, clothed in only short pants, entered a climatic chambermaintained at 32.5±0.5° C. with a relative humidity of 50%. About 1.5hours after entry into the chamber, a local cooling protocol wasinitiated with water-perfused stimulators placed on the head, chest,abdomen, or thigh. The subjects felt cooling of the face and thigh wasmore effective than cooling of the chest and abdomen in reducing theheat discomfort.

Essick et al.[Site-dependent and subject-related variations in perioralthermal sensitivity. Somatosensory Motor Research 21, 159-75, 2004]measured the thresholds for detecting cooling and cold pain on varioussites of the face, ventral forearm, and scalp for 34 young adults. Onthe face, the most sensitive sites were on the vermilion, which coulddetect a temperature change of about 0.5° C., followed by areas aroundthe mouth (the upper and lower hairy lip, mouth corner) and lateralchin. The mid-cheek and periauricular skin were less sensitive (able todetect a temperature change of about 2° C.), and the forearm and scalpwere least sensitive (able to detect a temperature change of about 3°C.). Essick et al. did not examine the areas around the nostrils.

In an earlier study (Wei, U.S. Ser. No. 14/544,355), DIPA-1-7, the mostpotent compound for dynamic cooling, was tested at other topical siteson the cranium. A 20 mg/mL solution was applied, using a cotton wipe,onto the skin above the buccal cheek, the parotid-masseteric cheek,temple, and the skin above the periauricular region, and the posteriormandible using the appropriate craniometric points (pterion, coronion,condylion, and gonion, respectively) as landmarks. Surprisingly, at allof these sites, other than the buccal cheek, little cooling wasobserved. Mild cooling was observed on the buccal cheek forapproximately 30 minutes, but this effect may have been due to thespread of the solution onto the receptive field of the infraorbitalnerve. DIPA-1-7 and related analogs were, however, potently active onthe eyelid margins.

To ascertain the sites and mechanisms of mask discomfort, weexperimented. In South Korea, DIPA-1-7 is available as a 30 gdermatological gel formulated as 1.5% wt/vol for the treatment of itch.This gel, called Intrinsic IB, was topically applied to different sitesof the face of five male volunteers (aged 32 to 56). The gel was appliedwith a forefinger at 0.03 to 0.05 mL to discrete sites of the facenormally covered by a mask: namely, 1. Alar crease. 2. External nares.3. Lateral cheek. 4. Nasolabial folds. 5. Philtrum. 6. Vermilion, and 7.Chin, as shown in FIG. 6. The Intrinsic IB was weighed before and aftereach application to record the amount that was applied. Surprisingly,the rank order of sensitivity was the same for all five subjects. Forcoolness and cold, the rank order was alar crease>external naresphiltrum>vermilion≅nasolabial folds>chin>>lateral cheek. The rank orderis most likely determined by the TRPM8 nerve fiber density and by thethickness of the skin at the site. The number of cell layers on thecheek is about 10±3, and 7 for the nasolabial folds (Zhen et al. Numberof cell layers of the stratum corneum in normal skin—relationship to theanatomical location on the body, age, sex and physical parameters. ArchDermatol Res (1999) 291: 555-559).

The subjects were then instructed to repeat the application but thistime to also use a N95 FFR for ˜1 hr. Afterwards the subjects were askedwhat was the best site to counteract the discomfort caused by the mask.Surprisingly and unexpectedly, although all sites gave coolness and coldsensations at various intensities, the test subjects stated that onlygel application on the external nares prevented the development of facemask discomfort. Applications to other sites, for example, to thenasolabial folds or lips did not relieve discomfort. If there was maskdiscomfort the application to the skin of the external nares (nostrils)was instantly effective. There was no ambiguity in the results for thesefive subjects. Everybody agreed that the gel on the external naresprevented or counteracted the mask discomfort, and this site ofapplication gave the best results.

FIG. 5. is an illustration of the human face showing the test sites forapplying a gel containing 1.5% (15 mg/mL) of DIPA-1-7. The sites areidentified by the numbered circles:

1. Alar crease. 2. External nares. 3. Lateral cheek. 4. Nasolabialfolds. 5. Philtrum. 6. Vermilion. 7. Chin. A 0.03 to 0.5 mL aliquot of acommercial gel containing 1.5% of DIPA1-7 (Intrinsic IB, Dong WhaPharmaceuticals, Seoul, Republic of Korea) was applied with a forefingerto the designated site.

Further discussion with the test subjects illuminated the mechanism ofdrug action. They said that application on the nasolabial folds or thelips give overt sensations of cold, but did not help much on the maskdiscomfort. On the other hand, the gel about the nares gave a sense offree, unimpeded cool airflow. The subjects no longer noticed that theywere wearing a mask and could, for example, continue to work on thecomputer without annoyance. The test subjects stated that if the nosefelt cold, air movement with each breath also felt fresh and cold, andthe resistance to breathing disappeared. This refreshed breathing wasnot present if the skin felt cold on the nasolabial folds or the chin.

In modern sensory physiology, the mechanism of action of TRPM8 agonistson mask discomfort has a distinct language. The skin of the nose tip,especially around the nostrils, has sensors for heat abstraction whenthe temperature drops to the cool range of <25° C. Activation of these“thermistors” or TRPM8 nerve endings on a cold day or the ski slopegives the subject a “runny nose.” The sensors convey the pleasure ofbreathing cool air by the seaside or on a breezy day. The sensors shutoff behind a face mask when airflow diminishes or occlude, and heataccumulates >25° C. There is no air movement. As noted earlier, in thenatural state, the sensors respond best to the temperature rate change,not static temperature. That is, the brain “sees” −Δ ° C./t and notabsolute ° C. A TRPM8 receptor agonist, such as DIPA-1-9 on the skin ofthe nostrils, restores and enhances the thermosensitivity to coolness.The air-conditioning system re-adjusts and the subject feels better witha sense of cool airflow when breathing. The subject forgets theannoyance of the facial covering.

This result also explains why masks are uncomfortable. Constantbreathing of static warm air causes discomfort, not just heat orhumidity. Refreshed breathing is a dynamic event. With a mask, thesubject feels suffocated because the air does not move. With cooling,there is a relief of discomfort. Sensory physiologists know that dynamicand not static neuronal discharges into the brain account for the heatabstraction sensation of cooling in humans. Refreshing cooling is adynamic event that requires the discharge of TRPM8 nerve fibers. DIPAcompounds on the nostrils accelerate this neuronal event.

Study 5 Methods of Delivery and Test Results

To further characterize this discovery, a method for selective drugdelivery to the skin of the external nares was improvised. The hollow ofthe volar palm forms a receptacle that holds about 0.5 to 1 mL ofliquid. This hollow is a perfect fit for the nose tip. An ideal deliverymethod to the external nares skin is to place a solution into the hollowand immerse the nose tip into the hollow for ˜5 sec. This techniqueallows an even distribution of drugs and comparisons of molecularpotency and duration of action. For example, to construct dose-responsedata, a TRPM8 agonist solution was stored at a fixed volume in adisposable reservoir unit (3 mL polyethylene bottle) at 0.25 to 4 mg/mLsolution in saline. Next, 0.08 mL aliquot is weighed and put onto thehollow of the palm. The nose tip is immersed into the hollow at a ˜45°angle for 5 sec, as shown in FIG. 7. The tip of the nose fits snuglyinto the palmar cavity and precisely delivers localized test solution tothe skin of the nostrils. Inhalation of the solution into the nasalcavity itself is optional but not necessary. Alternatively, one can usea gel in the hollow of the palm.

FIG. 7. is an illustration of a bioassay procedure of a cooling agentapplied to the external nares. The gray area is the palm hollow. Thestars on the nostrils are the contact points for the drug solution.

Surprisingly, this method of contacting a TRPM8 agonist yielded robustdose-response data, as shown in FIG. 8. In practice, a single localapplication of DIPA-1-9 at 2 mg/mL in saline is sufficient to overcomemask discomfort for at least 4 hr and can be repeated with equal effectfor another 4-hr period. By comparison, localized wiping of the DIPAanalogs to other facial areas, such as the upper lip, philtrum, lips,and chin, were less effective for reducing mask discomfort.

FIG. 8. is an illustration of the cooling effects of DIPA-1-9 applied tothe external nares using the hollow of the palm assay. The results werefor DIPA-1-9 at 0.25, 0.5, 1.0, 2.0, and 4.0 mg/mL dissolved in saline.

In summary, an essential part of this discovery is the recognition thatskin at the opening of the nostrils is highly sensitive to thermalstimuli and influences airflow comfort and discomfort. Wearing a maskbecomes uncomfortable when the ambient environment is >25° C. The secondpart of the discovery is the identification of selective TRPM8 agonistmolecules that can be precisely delivered to the nostril site to relievemask discomfort. The prior art does not record this method of selectivetopical delivery of a cooling agent to the external nares skin for thetreatment of mask discomfort.

REFERENCES

Publications are cited herein to more fully describe and disclose thediscovery and the state of the art to which the discovery pertains. Eachof these publications is incorporated herein by reference in itsentirety into the present disclosure.

1. A method for the treatment of sensory discomfort in a subject causedby the subject's wearing a facial mask covering for more than one hour,comprising: topically applying a liquid or semi-liquid composition tothe skin of the subject's external flares, the composition having atherapeutically effective amount of a compound with Formula 1 therein

wherein R is n-heptyl, n-octyl, or n-nonyl and wherein the compositionis carried by a delivery agent, the agent being adapted to deliver theFormula 1 compound when topically applied with the Formula 1 compoundthereby penetrating the skin of the external nares skin and beingtherapeutically effective to prevent, reduce, or eliminate the sensorydiscomfort while the subject continues to wear the mask.
 2. The methodas in claim 1 wherein the facial mask covering is a surgical mask,medical mask, or a procedure mask.
 3. The method as in claim 1 whereinthe facial mask covering is a N95 facial filtration respirator or asimilar filtration respirator unit.
 4. The method as in claim 1 whereinthe facial mask covering is an elastomeric respirator made of syntheticor natural rubber material.
 5. The method as in claim 1 wherein theliquid composition is a water or an isotonic saline solution.
 6. Themethod as in claim 1 wherein the composition is a rinse and has fromabout 0.01 to 0.025% by weight of the Formula 1 compound.
 7. The methodas in claim 1 wherein the semi-liquid composition is a gel or a cream.8. The method as in claim 1 wherein the composition has from about 0.05to 2% by weight of the Formula 1 compound.
 9. The method as in claim 1wherein the Formula 1 compound is 1-diisopropyl-phosphinoyl-heptane[DIPA-1-7], or diisopropyl-phosphinoyl-octane [DIPA-1-8], or1-diisopropyl-phosphinoyl-nonane [DIPA-1-9] and the therapeuticallyeffective amount per dose is from about 1 to 5 mg.
 10. A method for thetreatment of sensory discomfort in the nasal cavities of a subjectcaused by the subject's wearing a facial mask covering for more than onehour, comprising: providing a liquid or semi-liquid composition carriedby a delivery agent, the composition having a therapeutically effectiveamount of a compound with Formula 1 therein when the composition istopically applied so as to penetrate the skin of the external nares

wherein R is n-heptyl, n-octyl, or n-nonyl; and, instructing the subjectto topically apply a therapeutic dose of the composition to the skin ofthe subject's external nares, the therapeutical effect such that thesubject can continue to wear the facial mask with reduced or eliminatedsensory discomfort.
 11. The method as in claim 10 wherein the Formula 1compound is selected from the group consisting of1-diisopropyl-phosphinoyl-heptane [DIPA-1-7],diisopropyl-phosphinoyl-octane [DIPA-1-8], and1-diisopropyl-phosphinoyl-nonane [DIPA-1-9], and the therapeuticallyeffective amount per dose is from about 1 to 5 mg.
 12. The method as inclaim 10 wherein the reduced or eliminated sensory discomfort is for atleast 3 hours.